Visual colocalization of molecular species within sectioned tissue can provide insights into cellular biochemistry and can serve as the basis for further study of protein-organelle and protein-protein interactions. Visualization of abnormal protein coexpression in neoplastic tissue may elucidate components of oncogenic signaling pathways. Colocalization of COX2 and laminin-5, for example, has been observed at the cancer-stromal interface of lung adenocarcinoma and may be associated with abnormalities in p53 expression (1). Additionally, colocalization of β-catenin with compartment-specific markers revealed prognostic significance that was not found using traditional single stain methods (2).
Current colocalization methods, however, have several limitations. The most commonly used method, multi-color immunofluorescence (MCIF), is limited by the number of viable combinations of available fluorescent tags and can be negatively affected by spectral bleed-through, antibody cross-reactivity, photo-bleaching, and autofluorescence of paraffin-embedded tissue (3). Because of these limitations, this method is usually restricted to the simultaneous visualization of just two antigens plus a nuclear counterstain. Peroxidase or alkaline phosphatase-linked multicolor immunostaining is possible, but the maximum number of chromogenic substrates that can be differentiated within a single section is typically two or three (4). Moreover, multiple chromogenic substrates do not allow colocalization in the same cellular compartment due to the obscuring of lighter colors by darker chromogens. Additionally, the use of more than one primary antibody from the same species (i.e., mouse or rabbit) is usually impossible due to secondary antibody cross-reactivity.
A recent report of a multiplex immunostaining chip demonstrated simultaneous staining of a large section with a grid of many different antibodies, but since different portions of the tissue section are stained with each probe no colocalization information is obtained (5).
There is a long felt need in the art for compositions and method useful for detecting and visualizing multiple antigens in a sample such as a cell or tissue sample. The present invention satisfies these needs.